2-amino-5-(2-hydroxyethyl)-6-methyl-4(3H)-pyrimidinone

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

9 October 2017 - 12 October 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

EU-Method B.46 resp.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

2-amino-5-(2-hydroxyethyl)-6-methyl-4(3H)-pyrimidinone

Cas Number:

6940-45-0

Molecular formula:

C7H11N3O2

IUPAC Name:

2-amino-5-(2-hydroxyethyl)-6-methyl-4(3H)-pyrimidinone

impurity 1

Reference substance name:

Sodium acetate

EC Number:

204-823-8

EC Name:

Sodium acetate

Cas Number:

127-09-3

Molecular formula:

C2H4O2.Na

IUPAC Name:

sodium acetate

additive 1

Reference substance name:

Silicon dioxide

EC Number:

231-545-4

EC Name:

Silicon dioxide

Cas Number:

7631-86-9

Molecular formula:

SiO2

IUPAC Name:

Silicon dioxide

Test material form:

solid: granular

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 103901
- Expiration date of the lot/batch: 1 February 2019

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: The test item was stored in the test facility in a closed vessel at room temperature (20 ±5°C).
- Stability under test conditions: Stable, solid

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Vehicle:

unchanged (no vehicle)

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Tissue Replicate: 3
- Amount(s) applied (volume or weight with unit):
1: 25.6 mg
2: 24.7 mg
3: 25.8 mg

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30E-6 L

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30E-6 L
- Concentration (if solution): 5%

Duration of treatment / exposure:

~ 25 hours

Duration of post-treatment incubation (if applicable):

19 hours

Number of replicates:

3

Test system

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit):
- Concentration (if solution):

Tissue Replicate: test item Amount
1: 25.6 mg
2: 24.7 mg
3: 25.8 mg

VEHICLE
- Amount(s) applied (volume or weight with unit):
- Concentration (if solution):
- Lot/batch no. (if required):
- Purity:

NEGATIVE CONTROL
- Amount(s) applied (volume or weight):
30E-6 L
- Concentration (if solution):

POSITIVE CONTROL
- Amount(s) applied (volume or weight):
30E-6 L
- Concentration (if solution):

Duration of treatment / exposure:

~ 25 hours

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Value:

96

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: Based on visual inspection it was consluded that there was no damage on the test system.
- Direct-MTT reduction: In a pre-test it was demonstrated that no direct MTT reduction by the test item occurred; hence no data correction was necessary.
- Colour interference with MTT: In a pre-test it was demonstrated that the test item does not develop a colour without MTT addition

DEMONSTRATION OF TECHNICAL PROFICIENCY: Technical proficiency was demonstrated in Annex V.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes
- Range of historical values if different from the ones specified in the test guideline: n.a.

Any other information on results incl. tables

Designation	SP02 (trade name: 5OHMlC)	Positive Control
% Tissue viability (tissue 1)	97.0%	1.6%
% Tissue viability (tissue 2)	93.3%	1.6%
% Tissue viability (tissue 3)	97.7%	1.8%
% Tissue viability (mean)	96.0%	1.7%
± SD of mean tissue viability (%)	2.3%	0.1%

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test item SP02 (trade name: 5OHMlC) is considered as non-irritant to skin. After the treatment, the mean value of relative tissue viability was reduced to 96.0%. This value is above the threshold for skin irritation (50%). The optical density of the negative control was well within the required acceptability criterion of 0.8 ≤ mean OD ≤ 2.8. The positive control has met the acceptance criterion too, for thus ensuring the validity of the test system. Variation within replicates was within the accepted range for negative control, positive control and test item (required: ≤ 18%). For these reasons, the result of the test is considered valid.

Executive summary:

This in vitro study evaluates the potential of SP02 (trade name: 5OHMlC) to evoke skin irritation in a reconstructed human epidermis (RhE) test method. It consists of a topical exposure of the neat test item to a RhE model followed by a cell viability test. Cell viability is measured by dehydrogenase conversion of MTT 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromid, present in cell mitochondria, into a blue formazan salt that is quantitatively measured after extraction from tissues. The skin irritation potential is determined from the percentage reduction of cell viability in comparison to untreated negative controls.

Three tissues of the human skin model EpiDermTM were treated with SP02 for 60 minutes. The test item was applied directly to each tissue and spread to match the tissue size (0.63 cm2; as indicated by the supplier). DPBS-buffer was used as negative control and 5% SDS solution was used as positive control. After treatment with the negative control, the mean absorbance value was 1.9 (mean OD), falling within the required acceptability criterion: 0.8 ≤ mean OD ≤ 2.8. The positive control showed clear irritating effects. The mean value of relative tissue viability was reduced to 1.7% (required: ≤ 20%). The variation within the tissue replicates of negative control, positive control and test item was acceptable (required: ≤ 18%). After the treatment with the test item, the mean value of relative tissue viability was reduced to 96.0 %. This value is larger than the threshold for skin irritation potential (50%). Test items that induce values above the threshold of 50% are considered non-irritant to skin. Therefore, SP02 (trade name: 5OHMlC) is considered non-irritant to skin in the Reconstructed human Epidermis (RhE) Test Method.